Development of an efficient protocol for doubled haploids in pearl millet using anther culture

Doubled haploid (DH) production technology has been a valuable tool for the development of homozygous lines through anther culture within a short period in comparison with conventional breeding methods. Doubled haploid production in pearl millet through anther culture helps to overcome inbreeding depression which occurs due to continuous selfing for homozygous line development. We developed an efficient method to develop DH plants through anther culture in pearl millet in large numbers, which is the first report in pearl millet. The method encompasses a selection of the right stage of mother plant, optimal culture media, and influence of temperature for treatment of anther for callus induction followed by plant regeneration and flow cytometric analysis for ploidy determination. Anther culture experiments were done in proprietary pearl millet genotype of Rasi seeds '1988F1' (R x R cross) for achieving better calli or embryo induction and green plant regeneration in pearl millet. A significant frequency of 35.4% calli-embryoid response in Doubled-haploid Pearl millet Media (DPM4) was achieved at incubation temperature of 32 degrees C for spikes pre-treatment and anther initiation. This response was further improved to 44.4% by separating anthers from spikes on fourth day of incubation. Alteration of basal salt concentration and inclusion of 12% maltose as carbon source in calli induction media resulted in a higher number of embryo-like structures (ELS) which eventually increased the regeneration up to 12.9% in Regeneration Media (RM3). These experiments resulted in a promising procedure to develop DH plants in pearl millet to minimize the timeline for pure line development and trait introgression to accelerate breeding program.